Pursuant to 37 C.F.R. 1.71(e), Applicants note that a portion of this disclosure contains material which is subject to copyright protection. The copyright owner has no objection to the facsimile reproduction by anyone of the patent document or patent disclosure, as it appears in the Patent and Trademark Office patent file or records, but otherwise reserves all copyright rights whatsoever.
This invention pertains to the shuffling of nucleic acids to achieve or enhance mycotoxin detoxification, especially in plants.
xe2x80x9cMycotoxinsxe2x80x9d generically refer to a number of toxic molecules produced by fungal species, such as polyketides (including aflatoxins, demethylsterigmatocystin, O-methylsterigmatocystin etc.), fumonisins, alperisins (e.g., A1, A2, B1, B2), sphingofungins (A, B, C and D), trichothecenes, fumifungins, and the like. Polyketides are a large structurally diverse class of secondary metabolites synthesized by bacteria, fungi, and plants and are formed by a polyketide synthase (PKS) through the sequential condensation of small carboxylic acids. Katz and Donandio (1993) Annu Rev. Microbiool. 47:875-912; Brown et al. (1996) PNAS 93:14873-14877; Silva et al. (1996) J. Biol Chem. 271: 13600-608.
Aflatoxin B1, is the principal member of the aflatoxin (AF) family of polyketide mycotoxins produced by Aspergillus parasiticus, Aspergillus flavus and Aspergillus nomius. Aflatoxin B1 is the most potent mycotoxin known to man. For example, AF was characterized as the causative agent for the death of more than a hundred thousand poultry in England that had ingested AF-contaminated peanut meal. This discovery led to legislation regulating the trade of AF-contaminated agricultural commodities.
Sterigniatocystin (ST) is a related polyketide mycotoxin, which is produced by several members of the Aspergillus. ST is the second to last intermediate in the biosynthesis of AF. Kelkar et al. (1997) J. Biol Chem. 272: 1589-94. Various Aspergillus species that produce AF and ST are known to be pathogenic to corn, grains and nuts and are known to produce these mycotoxins during the growth of the crops and during storage, leading to the introduction of AF and ST into primary food stuffs. AF and ST are acutely toxic and carcinogenic and are a serious concern from human and animal health perspective. Busby and Wogan (1985) in Chemical Carcinogens (Searle ed., 1985) pp 945-1136, American Chemical Society, Washington D.C.
Trichothecenes are another family of sesquiterpenoid mycotoxins produced by Fusarium species and other molds that are known plant pathogens. These compounds are potent inhibitors of protein synthesis in eukaryotes (Kimura et al. (1998) J. Biol Chem. 273: 1654-1661) and reportedly bind to the 60S ribosomal subunits to prevent polypeptide chain initiation or elongation. Trichothecenes are also an important group of mycotoxins that cause serious problems of food pollution. They have been implicated in incidents of mycotoxicosis including vomiting, dermatitis and hemorrhagic septicemia in humans and livestock, resulting in loss of productivity and even death. Lastly, fumonisins (F) are another structurally distinct class of mycotoxins produced by several Fusarium species that is involved in food poisoning and toxic effects. Scott (1993) International Journal of Food Microbiology 18:257-270 and the references therein provide a review of the Fuminosins.
Thus, the contamination of corn, grains and nuts with various types of mycotoxins produced by pathogenic species such as Aspergillus and Fusarium is a major health and food pollution problem, as well as causing reduction in crop yields by being toxic to infected plants. These mycotoxins survive food processing, which adds to the problem. It is well known that ST and AF induce liver cancer and are linked to a specific mutation in a tumor suppressor gene. Brown et al. (1996) PNAS 93: 14873-14877. Natural aflatoxins and other mycotoxins like ST do not pose a major health threat per se; however, renal and hepatic oxidative detoxification of these compounds in contaminated foods by cytochrome P450 enzymes yields an epoxide which is cytotoxic.
For example, AFB1 is converted to its 15,16-exo-epoxide, which is a highly toxic mutagen. Silva et al. (1996), supra and references therein. It has been shown that this epoxide targets guanine residues and selectively alkylates the N-7 position of this purine in double-stranded DNA. Depurination of the alkylated base has been correlated to bladder cancer in laboratory mice, teratogenic effects in chicken embryos and liver cancer in humans. A direct correlation between DNA damage and human cancer has been established and is related to the mutational hot spots of p53, an important tumor suppressor gene. Approximately 50% of all cancers have associated altered p53 sequences.
Trading of AF-contaminated agricultural commodities is tightly regulated at both national and international levels. Compliance to these regulations causes the loss of millions of dollars in agricultural produce in US each year. Trade sanctions and health effects on mycotoxin contaminated grains add significantly to the losses (Brown et al. (1996) PNAS 93: 14873-14877).
Accordingly, it is highly desirable to transform various mycotoxins produced by fungal pathogens in various crops into inactive compounds with respect to plant, human and animal toxicity. This would alleviate important food pollution problems, as well as cost associated with complying with detecting AF-contamination in various crop commodities and destroying them. Surprisingly, the present invention provides for the detoxification of mycotoxis by transformation of the mycotoxins into non-toxic compounds. This detoxification is particularly useful in crops, thereby solving each of the problems outlined above, as well as providing a variety of other features which will be apparent upon review.
In the present invention, DNA shuffling is used to generate new or improved mycotoxin detoxification genes. These mycotoxin detoxification genes are used to provide enzymes which degrade mycotoxins, in agricultural and industrial processes. These new and/or improved genes have surprisingly superior properties as compared to naturally occurring mycotoxin detoxification genes.
In the methods for obtaining mycotoxin resistant genes, a plurality of parental forms (homologs) of a selected nucleic acid are recombined. The selected nucleic acid is derived either from one or more parental nucleic acid(s) which encodes an enzyme which degrades or modifies a mycotoxin, or a fragment thereof, or from a parental nucleic acid which does not encode mycotoxin detoxification, but which is a substrate for DNA shuffling to develop monooxygenase activity. The plurality of forms of the selected nucleic acid differ from each other in at least one (and typically two or more) nucleotides, and, upon recombination, provide a library of recombinant mycotoxin detoxification nucleic acids. The library can be an in vitro set of molecules, or present in cells, phage or the like. The library is screened to identify at least one recombinant mycotoxin detoxification nucleic acid that exhibits distinct or improved mycotoxin detoxification activity (typically in an encoded polypeptide) compared to the parental nucleic acid or nucleic acids.
In selecting for mycotoxin detoxification activity, a candidate shuffled DNA can be tested for encoded mycotoxin detoxification activity in essentially any process. Common processes that can be screened include screening for inactivation or modification of an aflatoxin, inactivation or modification of a sterigmatocystin, inactivation or modification of a trichothecene, and inactivation or modification of a fumonisin. Similarly, instead of, or in addition to, testing for an increase in mycotoxin detoxification activity, it is also desirable to screen for shuffled nucleic acids which produce higher levels of a mycotoxin detoxification nucleic acid or enhanced or reduced recombinant mycotoxin detoxification polypeptide expression, or increased stability encoded by the recombinant mycotoxin resistant nucleic acid.
A variety of screening methods can be used to screen a library, depending on the mycotoxin detoxification activity for which the library is selected. By way of example, the library to be screened can be present in a population of cells. The library is selected by growing the cells in or on a medium comprising the mycotoxin to be degraded and selecting for a detected physical difference between, e.g., oxidized or reduced forms of the mycotoxin and the non-oxidized or reduced form of the mycotoxin, either in the cell, or the extracellular medium. Alternately, survival of library cells on a medium which includes a mycotoxin can be used to screen the library.
Iterative selection for mycotoxin detoxification nucleic acids is also a feature of the invention. In these methods, a selected nucleic acid identified as encoding mycotoxin detoxification activity can be shuffled, either with the parental nucleic acids, or with other nucleic acids (e.g., mutated forms of the selected nucleic acid) to produce a second shuffled library. The second shuffled library is then selected for one or more form of mycotoxin detoxification activity, which can be the same or different than the mycotoxin detoxification activity previously selected.
This process can be iteratively repeated as many times as desired, until a nucleic acid with optimized or desired mycotoxin detoxification properties is obtained. If desired, any nucleic acid identified by any of the methods herein can be cloned and, optionally, expressed. Because of the need to reduce mycoxin pollution/contamination of foods, it is desirable to express mycotoxin detoxification nucleic acids in, e.g., plants, thereby reducing the occurrence of mycotoxins in the plants. Furthermore, mycotoxin detoxification in plants also adds to the vigor of the plants.
The invention also provides methods of increasing mycotoxin detoxification activity by whole genome shuffling. In these methods, a plurality of genomic nucleic acids are shuffled in a cell (in whole cell shuffling, entire genomes are shuffled, rather than specific sequences, although xe2x80x9cspikingxe2x80x9d of selected nucleic acids can be used to bias shuffling outcomes). The resulting shuffled nucleic acids are selected for one or more mycotoxin detoxification traits. The genomic nucleic acids can be from a species or strain different from the cell in which activity is desired. Similarly, the shuffling reaction can be performed in cells using genomic DNA from the same or different species, or strains. Strains or enzymes exhibiting enhanced activity can be identified.
The distinct or improved activity encoded by a nucleic acid identified after shuffling can encode one or more of a variety of properties, including, e.g., inactivation or modification of a polyketide, an aflatoxin, inactivation or modification of a sterigmatocystin, inactivation or modification of a trichothecene, inactivation or modification of a fumonisin, an increased ability to chemically modify a mycotoxin, an increase in the range of mycotoxin substrates which the distinct or improved nucleic acid operates on, an increased expression level of a polypeptide encoded by the nucleic acid, a decrease in susceptibility of a polypeptide encoded by the nucleic acid to protease cleavage, a decrease in susceptibility of a polypeptide encoded by the nucleic acid to high or low pH levels, a decrease in susceptibility of the protein encoded by the nucleic acid to high or low temperatures, and a decrease in toxicity to a host cell of a polypeptide encoded by the selected nucleic acid.
The selected nucleic acids to be shuffled can be from any of a variety of sources, including synthetic or cloned DNAs. Exemplar targets for recombination include: nucleic acids encoding a monooxygenase, a P450, trichothecene-3-O-acetyltransferase, a 3-O-Methyltransferase, a glutathione S-transferase, an epoxide hydrolase, an isomerase, a macrolide-O-acytyltransferase, a 3-O-acytyltransferase, and a cis-diol producing monooxygenase which is specific for furan. Typically, shuffled nucleic acids are cloned into expression vectors to achieve desired expression levels.
One feature of the invention is the production of libraries and shuffling mixtures for use in the methods as set forth above. For example, a phage display library comprising shuffled forms of a nucleic acid is provided. Similarly, a shuffling mixture comprising at least three homologous DNAs, each of which is derived from a nucleic acid encoding a polypeptide or polypeptide fragment, is provided. These polypeptides can be, for example, any of those noted herein.
Isolated nucleic acids identified by selection of the libraries in the methods above are also a feature of the invention, as are kits comprising any of: mycotoxin detoxification nucleic acid libraries, shuffled mycotoxin detoxification nucleic acids, instructional materials for practicing any of the methods herein, containers for holding other kit components, and the like.
Not Applicable.
Unless clearly indicated to the contrary, the following definitions supplement definitions of terms known in the art.
A xe2x80x9crecombinant monooxygenase nucleic acidxe2x80x9d is a recombinant nucleic acid encoding a protein or RNA which confers monooxygenase activity to a cell when the nucleic acid is expressed in the cell.
A xe2x80x9crecombinantxe2x80x9d nucleic acid is a nucleic acid produced by recombination between two or more nucleic acids, or any nucleic acid made by an in vitro or artificial process. The term xe2x80x9crecombinantxe2x80x9d when used with reference to a cell indicates that the cell comprises (and optionally replicates) a heterologous nucleic acid, or expresses a peptide or protein encoded by a heterologous nucleic acid. Recombinant cells can contain genes that are not found within the native (non-recombinant) form of the cell. Recombinant cells can also contain genes found in the native form of the cell where the genes are modified and re-introduced into the cell by artificial means. The term also encompasses cells that contain a nucleic acid endogenous to the cell that has been artificially modified without removing the nucleic acid from the cell; such modifications include those obtained by gene replacement, site-specific mutation, and related techniques.
A xe2x80x9crecombinant mycotoxin detoxification nucleic acidxe2x80x9d is a recombinant nucleic acid encoding a protein or RNA which confers mycotoxin detoxification or degradation activity to a cell when the nucleic acid is expressed in the cell (and, most typically, translated into a polypeptide).
A xe2x80x9cplurality of formsxe2x80x9d of a selected nucleic acid refers to a plurality of homologs of the nucleic acid. The homologs can be from naturally occurring homologs (e.g., two or more homologous genes) or by artificial synthesis of one or more nucleic acids having related sequences, or by modification of one or more nucleic acid to produce related nucleic acids. Nucleic acids are homologous when they are derived, naturally or artificially, from a common ancestor sequence. During natural evolution, this occurs when two or more descendent sequences diverge from a parent sequence over time, i.e., due to mutation and natural selection. Under artificial conditions, divergence occurs, e.g., in one of two ways. First, a given sequence can be artificially recombined with another sequence, as occurs, e.g., during typical cloning, to produce a descendent nucleic acid. Alternatively, a nucleic acid can be synthesized de novo, by synthesizing a nucleic acid which varies in sequence from a given parental nucleic acid sequence.
When there is no explicit knowledge about the ancestry of two nucleic acids, homology is typically inferred by sequence comparison between two sequences. Where two nucleic acid sequences show sequence similarity it is inferred that the two nucleic acids share a common ancestor. The precise level of sequence similarity required to establish homology varies in the art depending on a variety of factors. For purposes of this disclosure, two sequences are considered homologous where they share sufficient sequence identity to allow direct recombination to occur between two nucleic acid molecules (as opposed to recombination using oligonucleotide intermediates, which does not require sequence similarity to acheive recombination). Typically, nucleic acids require regions of close similarity spaced roughly the same distance apart to permit recombination to occur. The recombination can be in vitro or in vivo.
The terms xe2x80x9cidenticalxe2x80x9d or percent xe2x80x9cidentity,xe2x80x9d in the context of two or more nucleic acid or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same, when compared and aligned for maximum correspondence, as measured using one of the sequence comparison algorithms described below (or other algorithms available to persons of skill) or by visual inspection.
The phrase xe2x80x9csubstantially identical,xe2x80x9d in the context of two nucleic acids or polypeptides (e.g., DNAs encoding a monooxygenase, or the amino acid sequence of the monooxygenase) refers to two or more sequences or subsequences that have at least about 60%, preferably 80%, most preferably 90-95% nucleotide or amino acid residue identity, when compared and aligned for maximum correspondence, as measured using one of the following sequence comparison algorithms or by visual inspection. Such xe2x80x9csubstantially identicalxe2x80x9d sequences are typically considered to be homologous. Preferably, the xe2x80x9csubstantial identityxe2x80x9d exists over a region of the sequences that is at least about 50 residues in length, more preferably over a region of at least about 100 residues, and most preferably the sequences are substantially identical over at least about 150 residues, or over the full length of the two sequences to be compared.
For sequence comparison and homology determination, typically one sequence acts as a reference sequence to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters.
Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith and Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman and Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson and Lipman, Proc. Nat""l. Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by visual inspection (see generally, Ausubel et al., infra).
One example of algorithm that is suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm, which is described in Altschul et al., J. Mol. Biol. 215:403-410 (1990). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/). This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always  greater than 0) and N (penalty score for mismatching residues; always  less than 0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) of 10, a cutoff of 100, M=5, N=xe2x88x924, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1989) Proc. Natl. Acad. Sci. USA 89:10915).
In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul (1993) Proc. Nat""l. Acad. Sci. USA 90:5873-5787). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.
Another indication that two nucleic acid sequences are substantially identical/homologous is that the two molecules hybridize to each other under stringent conditions. The phrase xe2x80x9chybridizing specifically to,xe2x80x9d refers to the binding, duplexing, or hybridizing of a molecule only to a particular nucleotide sequence under stringent conditions, including when that sequence is present in a complex mixture (e.g., total cellular) DNA or RNA. xe2x80x9cBind(s) substantiallyxe2x80x9d refers to complementary hybridization between a probe nucleic acid and a target nucleic acid and embraces minor mismatches that can be accommodated by reducing the stringency of the hybridization media to achieve the desired detection of the target polynucleotide sequence.
xe2x80x9cStringent hybridization conditionsxe2x80x9d and xe2x80x9cstringent hybridization wash conditionsxe2x80x9d in the context of nucleic acid hybridization experiments such as Southern and northern hybridizations are sequence dependent, and are different under different environmental parameters. Longer sequences hybridize specifically at higher temperatures. An extensive guide to the hybridization of nucleic acids is found in Tijssen (1993) Laboratory Techniques in Biochemistry and Molecular Biologyxe2x80x94Hybridization with Nucleic Acid Probes part I chapter 2 xe2x80x9cOverview of principles of hybridization and the strategy of nucleic acid probe assays,xe2x80x9d Elsevier, N.Y. Generally, highly stringent hybridization and wash conditions are selected to be about 5xc2x0 C. lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. Typically, under xe2x80x9cstringent conditionsxe2x80x9d a probe will hybridize to its target subsequence, but no to unrelated sequences.
The Tm is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. Very stringent conditions are selected to be equal to the Tm for a particular probe. An example of stringent hybridization conditions for hybridization of complementary nucleic acids which have more than 100 complementary residues on a filter in a Southern or northern blot is 50% formamide with 1 mg of heparin at 42xc2x0 C., with the hybridization being carried out overnight. An example of highly stringent wash conditions is 0.15M NaCl at 72xc2x0 C. for about 15 minutes. An example of stringent wash conditions is a 0.2xc3x97SSC wash at 65xc2x0 C. for 15 minutes (see, Sambrook, infra., for a description of SSC buffer). Often, a high stringency wash is preceded by a low stringency wash to remove background probe signal. An example medium stringency wash for a duplex of, e.g., more than 100 nucleotides, is 1xc3x97SSC at 45xc2x0 C. for 15 minutes. An example low stringency wash for a duplex of, e.g., more than 100 nucleotides, is 4-6xc3x97SSC at 40xc2x0 C. for 15 minutes. For short probes (e.g., about 10 to 50 nucleotides), stringent conditions typically involve salt concentrations of less than about 1.0 M Na ion, typically about 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3, and the temperature is typically at least about 30xc2x0 C. Stringent conditions can also be achieved with the addition of destabilizing agents such as formamide. In general, a signal to noise ratio of 2xc3x97 (or higher) than that observed for an unrelated probe in the particular hybridization assay indicates detection of a specific hybridization.
A further indication that two nucleic acid sequences or polypeptides are substantially identical/homologous is that the polypeptide encoded by the first nucleic acid is immunologically cross reactive with, or specifically binds to, the polypeptide encoded by the second nucleic acid. Thus, a polypeptide is typically substantially identical to a second polypeptide, for example, where the two peptides differ only by conservative substitutions.
xe2x80x9cConservatively modified variationsxe2x80x9d of a particular polynucleotide sequence refers to those polynucleotides that encode identical or essentially identical amino acid sequences, or where the polynucleotide does not encode an amino acid sequence, to essentially identical sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given polypeptide. For instance, the codons CGU, CGC, CGA, CGG, AGA, and AGG all encode the amino acid arginine. Thus, at every position where an arginine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are xe2x80x9csilent variations,xe2x80x9d which are one species of xe2x80x9cconservatively modified variations.xe2x80x9d Every polynucleotide sequence described herein which encodes a polypeptide also describes every possible silent variation, except where otherwise noted. One of skill will recognize that each codon in a nucleic acid (except AUG, which is ordinarily the only codon for methionine) can be modified to yield a functionally identical molecule by standard techniques. Accordingly, each xe2x80x9csilent variationxe2x80x9d of a nucleic acid which encodes a polypeptide is implicit in each described sequence.
Furthermore, one of skill will recognize that individual substitutions, deletions or additions which alter, add or delete a single amino acid or a small percentage of amino acids (typically less than 5%, more typically less than 1%) in an encoded sequence are xe2x80x9cconservatively modified variationsxe2x80x9d where the alterations result in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. The following five groups each contain amino acids that are conservative substitutions for one another: Aliphatic: Glycine (G), Alanine (A), Valine (V), Leucine (L), Isoleucine (I); Aromatic: Phenylalanine (F), Tyrosine (Y), Tryptophan (W); Sulfur-containing: Methionine (M), Cysteine (C); Basic: Arginine (R), Lysine (K), Histidine (H); Acidic: Aspartic acid (D), Glutamic acid (E), Asparagine (N), Glutamine (Q). See also, Creighton (1984) Proteins, W. H. Freeman and Company.
In addition, individual substitutions, deletions or additions which alter, add or delete a single amino acid or a small percentage of amino acids in an encoded sequence are also xe2x80x9cconservatively modified variations.xe2x80x9d Sequences that differ by conservative variations are generally homologous.
A xe2x80x9csubsequencexe2x80x9d refers to a sequence of nucleic acids or amino acids that comprise a part of a longer sequence of nucleic acids or amino acids (e.g., polypeptide) respectively.
The term xe2x80x9cgenexe2x80x9d is used broadly to refer to any segment of DNA associated with expression of a given RNA or protein. Thus, genes include regions encoding expressed RNAs (which typically include polypeptide coding sequences) and, often, the regulatory sequences required for their expression. Genes can be obtained from a variety of sources, including cloning from a source of interest or synthesizing from known or predicted sequence information, and may include sequences designed to have desired parameters.
The term xe2x80x9cisolatedxe2x80x9d, when applied to a nucleic acid or protein, denotes that the nucleic acid or protein is essentially free of other cellular components with which it is associated in the natural state.
The term xe2x80x9cnucleic acidxe2x80x9d refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides which have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g. degenerate codon substitutions) and complementary sequences and as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et al. (1991) Nucleic Acid Res. 19: 5081; Ohtsuka et al. (1985) J. Biol. Chem. 260: 2605-2608; Cassol et al. (1992); Rossolini et al. (1994) Mol. Cell. Probes 8: 91-98). The term nucleic acid is generic to the terms xe2x80x9cgenexe2x80x9d, xe2x80x9cDNA,xe2x80x9d xe2x80x9ccDNAxe2x80x9d, xe2x80x9coligonucleotide,xe2x80x9d xe2x80x9cRNA,xe2x80x9d xe2x80x9cmRNA,xe2x80x9d and the like.
xe2x80x9cNucleic acid derived from a genexe2x80x9d refers to a nucleic acid for whose synthesis the gene, or a subsequence thereof, has ultimately served as a template. Thus, an mRNA, a cDNA reverse transcribed from an mRNA, an RNA transcribed from that cDNA, a DNA amplified from the cDNA, an RNA transcribed from the amplified DNA, etc., are all derived from the gene and detection of such derived products is indicative of the presence and/or abundance of the original gene and/or gene transcript in a sample.
A nucleic acid is xe2x80x9coperably linkedxe2x80x9d when it is placed into a functional relationship with another nucleic acid sequence. For instance, a promoter or enhancer is operably linked to a coding sequence if it increases the transcription of the coding sequence.
A xe2x80x9crecombinant expression cassettexe2x80x9d or simply an xe2x80x9cexpression cassettexe2x80x9d is a nucleic acid construct, generated recombinantly or synthetically, with nucleic acid elements that are capable of effecting expression of a structural gene in hosts compatible with such sequences. Expression cassettes include at least promoters and optionally, transcription termination signals. Typically, the recombinant expression cassette includes a nucleic acid to be transcribed (e.g., a nucleic acid encoding a desired polypeptide), and a promoter. Additional factors necessary or helpful in effecting expression may also be used as described herein. For example, an expression cassette can also include nucleotide sequences that encode a signal sequence that directs secretion of an expressed protein from the host cell. Transcription termination signals, enhancers, and other nucleic acid sequences that influence gene expression, can also be included in an expression cassette.